Project Summary Filarial nematodes currently infect over 150 million people worldwide, representing the leading cause of morbidity in the developing world. Current medications are inadequate for control and elimination of these parasitic infections, necessitating a better understanding of the basic biology of these worms and the development of new drugs and vaccines. A promising target for intervention is the relationship between filarial nematodes and their endosymbiotic bacteria Wolbachia. Elimination of the bacteria renders the worm unable to molt, develop, and reproduce; conversely, the bacteria cannot be cultured outside the worm. This indicates an obligate symbiotic relationship for which the mechanism remains largely unknown. Wolbachia populations remain relatively constant over the lifecycle of the parasite with the exception of large expansions during larval development from L3s to L4s and embryogenesis. Interestingly, these two stages are the points at which treatment with antibiotics has the most deleterious effects on the worms. It remains unknown, however, what the mechanisms of endosymbiosis are and if/how they differ over the lifecycle of the worm. To answer these questions, I first performed dual RNA-seq of both the nematode and the bacteria over the lifecycle of the parasite. I will use these data to build a co-expression network for the two organisms to identify co-expressed pathways necessary for mediating the endosymbiotic relationship. These analyses will provide potential pathways of interest that I will then query through manipulations of both worm development and bacterial expansion, making use of the in vitro culture system for the L3 to L4 larval molt of B. malayi. I will culture B. malayi with a cysteine protease inhibitor, which inhibits molting at various stages of the molt to determine if the cue for Wolbachia expansion comes from B. malayi and if so, at which stage. I will also culture B. malayi at several concentrations of antibiotics to determine which stage of molting requires bacterial products or cues. Together these experiments will allow me to characterize the molecular basis of the endosymbiotic relationship and determine at which stage of development reciprocal cues are required. Metabolic potential is increasingly being viewed as a critical element governing a pathogen's ability to survive in infected hosts. I hypothesize that the presence of Wolbachia, which likely provisions necessary cofactors and metabolites to B. malayi, influences the metabolic capacity of the worm and therefore its ability to survive in the human host. I will use genomic and transcriptomic data to reconstruct filarial and bacterial metabolism using flux balance analysis and compare it to that of Loa loa, a Wolbachia-free filarial nematode. Through determining the differing essential metabolic requirements of Wolbachia-containing and Wolbachia-free filarial worms, we will discover novel drug targets that are safe for use in L. loa co-endemic areas.